Four isoforms of protein phosphatase 1 (PP1, PP1, PP1? 1, and PP1?2), derived from three genes, are virtually identical, except at their extreme C-termini. The isoforms PP1?1 and mammalian-specific PP1?2 derive from splice-variant transcripts of a single gene (Ppp1cc). Although all four PP1 isoforms are expressed in testis, PP1?2 is by far predominant in male germ cells, and is the sole PP1 isoform present in mammalian spermatozoa. The PP1?2 isoform has a unique 22 amino acid C-terminal tail. Targeted deletion of Ppp1cc leads to sterility of -/- males due to grossly defective morphological development of spermatids and faulty spermiation. We tested whether transgenic expression of PP1?2 or PP1?1 driven by the testis- specific Pgk2 promoter or the Ppp1cc endogenous promoter could restore fertility of Ppp1cc -/- males (rescue mice). Our preliminary studies show that normal sperm morphogenesis and fertility are restored only when adequately high levels of PP1?2 are expressed in differentiating male germ cells. This restoration of fertility occurs even when the PP1?1 isoform is completely absent. Low levels of PP1?2 result in poorly motile sperm with several morphological abnormalities. PP1?1 rescue mice also produce greater sperm numbers than Ppp1cc -/- mice. However, sperm are grossly malformed and completely immotile. The focus of this proposal is to identify the absolute and isoform specific requirement for adequate levels of PP1?2 leading to normal structure and function of mammalian spermatozoa. We will determine whether the isoform specific function of PP1?2 is determined by its C-terminus. Finally, we will ascertain how limiting levels of PP1?2 alter the sperm proteome, thus resulting in structural deformities and impaired sperm function. These studies will continue to unravel novel mechanisms involved in mammalian sperm morphogenesis and function, and thus lead to simple diagnostic tests for fertility in man and other mammals based on the level of PP1?2 in spermatozoa. PUBLIC HEALTH RELEVANCE: Statement Alternatively spliced protein phosphatase PP1?2 isoform of PP1 ? gene plays an essential role in spermatogenesis and mammalian reproduction. However, molecular mechanisms responsible for its regulation and action in testis and spermatozoa are unclear. We will identify how PP1? gene and its protein product PP1?2 in testis and spermatozoa are responsible for sperm formation and function.